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March 2006
Data presented on the Certificate of Analysis (C of A) for biological indicators (BI) used
to monitor steam sterilization processes typically include the spore population, D-value(s)
determined at one or more temperatures and the Z-value.
Often, the end users of the BIs are operating their sterilizers at a temperature for which
the D-value is not reported on the C of A. A common question BI users ask is how can I
calculate the D-value and kill time of the BI when my sterilization temperature is not one
of those listed on the Certificate of Analysis?
To calculate the theoretical kill time of the BI at a specified temperature, two data points
are required; the initial population of the BI, and the D-value at the specified temperature.
This Spore News will address the methods for calculating the D-value and the theoretical
kill time in these situations. For the purpose of this discussion, we will not be using the
survival/kill time calculation formulas as presented in ISO and USP1. These formulas
add a safety factor into the calculations resulting in values that generally bracket the
empirically derived data. In other words, the calculated kill time value will be higher
than the empirically determined kill time.
Lets begin by calculating the theoretical kill time for a temperature that is listed on the C
of A. Data extracted from a C of A for a BI lot (Figure 1) shall be used in the following
examples.
1
Survival time/dose = not less than the D-value X (log10 labeled viable test organism count per carrier 2)
Kill time/dose = not more than the D-value X (log10 labeled viable test organism count per carrier + 4)
Z-value 15.2 C
D-value reproducible only when exposed in an AAMI BIER vessel and cultured under the exact conditions used to obtain results reported here. MPN and
Survivor Curve method used.
Units are manufactured in compliance with SGM Biotech's quality standards, USP, EN 866 and ISO 11138 guidelines and all appropriate subsections.
(1)
D-value calculated using the Limited Holcomb-Spearman-Karber method.
(2)
Survival/Kill values are calculated according to USP and ISO 11138.
(3)
Empirically derived data.
The theoretical kill time is a product of the log of the population (N0) plus one (1) and the
D-value.
The first step of this equation is to take the log of the initial population. It is often
assumed that this will be the spore log reduction (SLR) required to reduce the spore
population to zero surviving spores. This in fact is not true. This SLR will reduce the
spore population to an average of 100 or one (100 = 1) surviving spore per BI (see Figure
2). One is added to the SLR in the calculation to reduce the number of spores to less than
one per unit resulting in total kill of all test units.
From the C of A for lot S-308 (Figure 1), the Heat Shocked population (N0) is 2.6 x 106
per unit and the D121-value is 1.7 minutes. Substituting these values into the equation we
have:
Theoretical kill time = (Log10 2.6 x 106 + 1) x 1.7 minutes = 12.6 minutes
Population
3.4 2.6 X 10 5.2 X 10 1.00E+04
3 4 1.00E+03
5.1 2.6 X 10 5.2 X 10
1.00E+02
6.8 2.6 X 102 5.2 X 103 1.00E+01
8.5 2.6 X 101 5.2 X 102 1.00E+00
0 1
10.2 2.6 X 10 5.2 X 10 1.00E-01 0 2 4 6 8 10 12 14 16
-1 0
11.9 2.6 X 10 5.2 X 10 1.00E-02
-2 Minutes
13.6 2.6 X 10 5.2 X 10-1
Figure 2. An example of a Spore Log Reduction (SLR)
To determine the kill time for lot S-308, the first step will be to calculate the D131-value
using the correct Z-value. When the Z-value and the actual sterilization temperature are
not convenient numbers to work with mathematically, the following formula (derived
from the Z-value calculation formula presented in ISO 11138-3:1995(E) Annex B is
necessary:
To determine the kill time, the first step will be to calculate the D116.5-value using the
formula:
T 2 T1
D 1 = D 2 10 Z
To determine the kill time, the first step will be to calculate the D124-value using the
formula:
T 2 T1
D 1 = D 2 10 Z
121 124
D124 = 1 .7 10 15 .2
A single BI should be killed in approximately 8.2 minutes. During data collection on BIs
from lot S-308, the 8 minute exposure at 124C resulted in 17 units killed out of 20
exposed.
The D124-value was one of the D-values experimentally determined on lot S-308 and used
in the Z-value calculation. The D124-value was determined to be 1.0 minute and the 9
minute exposure resulted in total kill of all 20 BIs.
Discussion
Z-value curves for Geobacillus stearothermophilus spores tend to be concave downward
over a wide temperature range (i.e. the Z-values tend to decrease as the temperature
increases). The fact that resistometers (test vessels) perform most accurately at
temperatures below 130C certainly contributes to this situation.
For the BI manufacturer, ISO states that a Z-value shall be calculated from D-values
obtained in the temperature range of 110C-130C. SGM calculates the Z-value using D-
values obtained from three temperatures in the 120C-130C range.
Based on the ISO requirements and SGM current operating procedure for Z-value
determinations, this formula will result in a good estimation of D-values in the 110C-
130C temperature range using the Z-value printed on the C of A . This is supported by
comparing the empirically determined results with the calculated results as in examples 1
and 4.
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